Article Figures & Data
Figures
- FIGURE 1.
Intravital imaging maintains intact blood flow without damaging the pancreas. (A) Setup for intravital two-photon pancreas imaging. A heated suction window stabilizes the surgically exposed pancreas for imaging. (B and C) Representative maximum intensity projection images of islets imaged intravitally through the suction imaging window captured using two-photon microscopy. Vascular space is labeled with 70-kDa dextran-rhodamine (red). Images are representative of seven experiments. (B) Islets are identifiable by their dense convoluted vasculature compared with exocrine tissue vasculature. The border of the islet is identified with a yellow dashed line. Scale bar, 30 μm. (C) NOD mouse islet with transferred BDC-2.5 T cells (green). The collagen fluorescence is provided by the second harmonic (blue), which demonstrates that the T cell infiltration is inside the islet basement membrane. Scale bar, 100 μm. (D) Neutrophils do not accumulate at the site of imaging. Fluorescently labeled neutrophils were transferred into mice prior to surgical exposure and imaging of the pancreas through the suction window. The number of neutrophils was counted every 90 s. The lack of neutrophil accumulation shows that the imaging site was not damaged during imaging. Data are representative of one islet per mouse in three experiments. (E) Suction imaging window does not impede blood flow. Fluorescent beads were tracked within blood vessels of different diameter within and around the pancreatic islets. Each dot represents one bead. Data are representative of three islets per mouse in two experiments.
- FIGURE 2.
Explanted islet imaging allows high resolution, high throughput imaging. (A) Setup of explanted islet two-photon imaging. Isolated pancreatic islets were mounted in low melting temperature agarose and maintained at 35–37°C with constant flow of oxygenated media. (B) Representative multiple field image of explanted islets. BDC-2.5 T cells (red) were transferred into WT NOD recipients, and islets were isolated. Image shows a maximum intensity projection of explanted islets. Nuclei are labeled with Hoechst 33342 (blue). Scale bar, 200 μm. Images are representative of >3 experiments.
- FIGURE 3.
T cell motility increases with progression of islet infiltration. (A and B) Activated BDC-2.5 T cells (green) were fluorescently labeled and transferred 24 h prior to imaging. Representative maximum intensity projection images from explanted (A) or intravital (B) islets captured using two-photon microscopy. Dashed lines represent the islet border. Green lines represent 10-min paths of BDC-2.5 T cell movement. Images are representative of islets with mild infiltration (<30% of islet volume infiltrated) or advanced infiltration (30–60% of islet volume infiltrated). Scale bars, 50 μm. (C–E) Quantification of T cell motility within explanted or intravital islets. Data pooled from 16 explanted islets from four mice in three independent experiments and 16 intravital islets from seven mice in seven independent experiments. *p < 0.05, measured by Student t test. (C) Linear correlation of the average T cell velocity within an islet versus the percentage of the infiltrated islet volume. Each dot represents the average of all of the tracked T cells within a single islet. (D and E) Average of individual islets. (D) T cell crawling speed in explanted versus intravital islets. (E) Fold increase in crawling speed between islets with mild and advanced infiltration.
- FIGURE 4.
T cells reduce arrest and increase displacement as islet infiltration progresses. Activated BDC-2.5 T cells were fluorescently labeled and transferred 24 h prior to imaging, as in Fig. 3. Data were pooled from 16 explanted islets from four mice in three independent experiments and 16 intravital islets from seven mice in seven independent experiments. (A and B) Average of individual islets. (A) T cell track straightness (1 = cell moves in a straight line). (B and C) T cell arrest coefficient (% of time crawling speed is <2 μm/min). (D) Mean squared displacement (μm2) over time. *p < 0.05, **p < 0.01, ***p < 0.001; measured by two-way ANOVA with Bonferroni posttests or Student t test.
- FIGURE 5.
Early T cell arrest is Ag dependent. BDC-2.5 T cells were fluorescently labeled and transferred into WT NOD or NOD.C6 recipient mice 48 h prior to imaging to determine infiltration state. BDC-6.9 T cells and BDC-2.5 T cells were cotransferred 24 h prior to imaging to determine T cell motility. The Ag for BDC-6.9 T cells is absent in the NOD.C6 recipients. Data represent 25 WT islets from four mice in four experiments and 25 NOD.C6 islets from five mice in five experiments. Each point represents the average T cell motility within one islet. (A) In WT NOD islets in which the Ag was present for BDC-2.5 and BDC-6.9 T cells, both types of T cells increase motility at a similar rate as islet infiltration increases. (B) In NOD.C6 islets, in which the Ag is present for BDC-2.5 T cells, but absent for BDC-6.9 T cells, the BDC-6.9 T cells move faster in the absence of their Ag. (C) The ratio of average BDC-6.9 T cell motility to the average BDC-2.5 T cell motility within the same islet. Infiltration states: very mild (0–5%), mild (5–30%), and advanced (30–60%). (D) Comparison of the arrest coefficient of all BDC-2.5 and BDC-6.9 T cells within islets with mild infiltration. BDC-6.9 T cells (No Ag) have reduced arrest. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed Student t test.
- FIGURE 6.
Sustained T cell–CD11c+ APC interactions are lost with progression of islet infiltration. Fluorescently labeled BDC-2.5 T cells were transferred into CD11c-YFP hosts 24 h prior to islet isolation and imaging. Data represent 15 islets from five mice in five independent experiments. (A and B) Maximum intensity projection images showing BDC-2.5 (red) and CD11c+ APCs (green) within pancreatic islets. Yellow box indicates the region shown in time-lapse images on the right. Gray circles highlight the CD11c+ APCs that the T cell of interest has contacted; yellow arrows show current T cell–APC contacts. Time stamps = min:s. (A) Sustained T cell–APC interaction in an islet with mild infiltration. Scale bar, 40 μm for whole islet and 10 μm for time-lapse images. (B) Transient T cell contacts with different CD11c+ APCs in an islet with advanced infiltration. Scale bar, 50 μm for whole islet and 20 μm for time-lapse images. (C) Average percentage of T cells within individual islets that contact CD11c+ APCs for at least 2 min. (D) Average percentage of T cells that contacted CD11c+ APCs, which had sustained interactions of ≥10 min. (E) Duration of T cell– CD11c+ APC contacts. *p < 0.001, ***p < 0.001 by two-tailed Student t test.
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